In the early 1990s, there appeared anecdotal reports suggesting that the symptoms of multiple sclerosis (MS) were alleviated by drinking tea made from the plant Ruta graveolens. Various laboratory studies were then undertaken in efforts to identify the pharmacologically active component(s) of Ruta graveolens. It was determined that extracts from Ruta graveolens could block delayed-rectifier potassium currents in nodes of Ranvier (Bohuslavizki K. H. et al., 1994, Gen. Physiol. Biophys. 13: 309). On this basis, it was postulated that certain component(s) of Ruta graveolens blocked potassium currents in either neurons that had become demyelinated due to MS or in myelin-reactive lymphocytes. 5-Methoxypsoralen (5-MOP), a compound that has been clinically used in the treatment of psoriasis was identified as a component of Ruta graveolens that blocked Kv1.2 channels in neurons and Kv1.3 channels in T cells (Wulff et al., 1998, J. Med. Chem. 41: 4542). Subsequent single case studies indicated that the administration of 5-MOP alleviate the symptoms of MS in several patients with MS (Bohuslavizki K. H. et al., 1993, Neuroopthalmol. 13: 191). However, 5-MOP blocks potassium channels with a very low affinity (Kd=100 μM). Thus, there remained a need in the art for the synthesis and/or identification of new psoralen compounds that would inhibit potassium channels much more potently and thus provide therapeutic efficacy against MS and other autoimmune disorders.
Kv1.3 Channel in T Cells.
The predominant voltage-gated channel in human T-lymphocytes is encoded by Kv1.3, a Shaker-related gene. Kv1.3 has been characterized extensively at the molecular and physiological level and plays a vital role in controlling T-lymphocyte proliferation, mainly by maintaining the resting membrane potential of resting T-lymphocytes. Recently, encephalitogenic and arthritogenic rat T cells that were chronically activated with myelin antigens, were found to express a unique channel phenotype (high Kv1.3 channels and low IKCa1 channels), distinct from that seen in quiescent and acutely activated T cells (Beeton et al., 2001, Proc. Natl. Acad. Sci. USA 98:13942). In a subsequent study, we have confirmed this finding in myelin antigen specific T cells from patients with MS. Contrary to myelin-reactive T cells from healthy controls and to mitogen or control antigen activated T cells from MS patients, myelin reactive T cells from MS patients predominantly expressed surface markers of terminally differentiated effector memory T cells (CCR7−CD45RA−) and exhibited the Kv1.3high IKCa1low phenotype (Wulff et al., 2003, J. Clin. Invest. 111:1703). In the same study we could show that this special K+ channel phenotype made the proliferation of effector memory T cells highly sensitive to inhibition by Kv1.3 blockers. NaÏve and central memory T cells were only affected at 10-fold higher concentrations of Kv1.3 blockers and could escape Kv1.3 inhibition during subsequent stimulation through the up-regulation of the calcium-activated potassium channel IKCa1. It would be thus possible to target the disease-inducing effector memory T cell population without affecting the normal immune response. In a proof of this concept the Kv1.3-blocking peptide ShK has been recently shown to prevent and treat experimental autoimmune encephalomyelitis in Lewis rats, an animal model for MS (Beeton et al., 2001, Proc. Natl. Acad. Sci. USA 98:13942).
Kv1.3 and IKCa1 Expression and Functional Roles in Human Naïve and Memory T-Cells
Human T-cells are divided into three subsets, naïve, central memory (TCM) and effector memory T (TEM) cells, based on the expression of the chemokine receptor CCR7 and the phosphatase CD45RA. Naïve (CCR7+CD45RA+) and TCM (CCR7+CD45RA−) cells migrate to the lymph node using CCR7 as an entry code, before migrating to sites of inflammation. In contrast, TEM cells have the ability to home directly to sites of inflammation, where they can secrete high amounts of interferon (IFN)-γ and tumor necrosis factor (TNF)-α and exhibit immediate effector function. The expression patterns of Kv1.3 and IKCa1 change dramatically as naïve cells become memory cells. At rest, CD4+ and CD8+T-cells of all three subsets exhibit ˜200 to 400 Kv1.3 channels, and 0 to 30 IKCa1 channels (Wulff et al., 2003, J. Clin. Invest. 111:1703). Activation has diametrically opposite effects on channel expression; as naïve and TCM cells move from resting to proliferating blast cells, they transcriptionally up-regulate IKCa1 to ˜500 channels per cell. In contrast, activation of TEM cells enhances Kv1.3 expression without any change in IKCa1 levels (Wulff et al., 2003, J. Clin. Invest. 111:1703). Functional Kv1.3 expression increases dramatically within 15 h of activation to a level of 1500 Kv1.3 channels/cell, remains elevated for the following 48 to 72 h, and then returns to baseline over the next five days (Beeton et al., 2003, J. Biol. Chem. 278:9928)
The subset-specific channel expression has important functional consequences, since Kv1.3 and IKCa1 regulate Ca2+ entry into T-cells through Ca2+-release-activated Ca2+ (CRAC) channels that exhibit ‘upside-down’ voltage-dependence compared with voltage-gated Ca2+ channels. A negative membrane potential drives Ca2+ entry through CRAC channels. The electrochemical gradient supporting Ca2+ entry is initially large, resulting in significant Ca2+ influx. However, Ca2+ entry results in depolarization of the plasma membrane, limiting further influx. To maintain Ca2+ entry over the time scale required for gene transcription, a balancing cation efflux is necessary; this is provided by the efflux of K+ ions through Kv1.3 and/or IKCa1 channels, which supply the electrochemical driving force for Ca2+ entry via membrane hyperpolarization.
Depolarization resulting from Kv1.3 and IKCa1 blockade is inhibitory for Ca2+ influx, signaling and lymphocyte activation. As Kv1.3 channels predominate in resting T-cells of the three subsets, the Kv1.3 blocker ShK, but not the IKCa1 blocker TRAM-34, suppress antigen or mitogen-driven activation. However, ShK is 10-fold more effective on TEM cells than on naïve and TCM cells (IC50 values of 400 pM and 4 nM, respectively), due to the fact that the latter cells rapidly up-regulate IKCa1 after stimulation and become less sensitive to Kv1.3 inhibitors (Wulff et al., 2003, J. Clin. Invest. 111:1703). Once IKCa1 is up-regulated in naïve and TCM cells, the reactivation of these cells is sensitive to IKCa1 but not Kv1.3 blockade. Naïve and TCM cells can up-regulate IKCa1 following mitogen or antigen stimulation, even if their initial activation is suppressed by Kv1.3 blockade; and can consequently escape further inhibition by Kv1.3 inhibitors (Wulff et al., 2003, J. Clin. Invest. 111:1703). Early in vivo studies support these in vitro findings. The Kv1.3 blockers MgTX (Koo et al., 1997, J. Immunol. 158:1520) and correolide (Koo et al., 1999, Cell Immunol. 197:99) effectively suppress the primary delayed-type hypersensitivity (DTH) response in mini-pigs, but are much less effective in suppressing the secondary DTH response, presumably due to the fact that the activated naïve or TCM cells involved have up-regulated IKCa1 expression. In contrast, TEM cells exclusively up-regulate Kv1.3 channels, and are persistently suppressed by Kv1.3 inhibitors.